Abstract. Origin licensing is a prerequisite for eukaryotic DNA replication, and it occurs from the late M to early G1 phases of the cell cycle. During this process, all replication origins are licensed by chromatin binding of the MCM2-7 heterohexameric complexes. In the following S phase, licensed origins fire only once when chromatin-bound MCM2-7 complexes assemble active replicative helicases to unwind DNA. While only a small fraction (5~10%) of licensed origins are used to initiate bi-directional replication forks in the given replicon, all remaining origins are backups termed dormant origins. Such origins are activated for the recovery of stalled replication forks, contributing to the completion of DNA replication and tumor suppression in mice. However, it remains unknown if dormant origins also play a protective role in response to fork-stalling DNA damage induced by ultraviolet (UV) radiation, the major risk factor for skin cancers in humans. UV produces two major types of fork-stalling DNA lesions: cyclobutane pyrimidine dimmers (CPDs) and pyrimidine(6-4)pyrimidone photoproducts (6-4PPs). While nucleotide excision repair (NER) removes these lesions, CPDs can persist longer even in S phase to inhibit DNA synthesis by the replicative polymerases. To overcome this problem, eukaryotic cells instead utilize Pol?, a translesion synthesis (TLS) DNA polymerase, as it accurately replicates through (bypass) the vast majority of CPDs to suppress mutations and cancers. In contrast, much fewer 6-4PPs are left unrepaired. However, as TLS across 6-4PPs can be highly error-prone, residual 6-4PPs must be bypassed by alternative, error-free mechanisms to avoid cancer-initiating mutations. We hypothesize that stalled fork rescue by dormant origins promotes error-free bypass of 6-4PPs. Dormant origin firing sustains fork progression, making undamaged sister-chromatids available as templates for the error-free mechanisms such as template switch and homologous recombination. This hypothesis is supported by recent published studies by others and our preliminary data on the Mcm4chaos3 (Mcm4c3) mouse model. Mcm4c3/c3 cells have a reduced number of dormant origins (~50% of wild-type level), displaying a rare type of UV-induced mutations termed triplet mutations. As evidence indicates that triplet mutations are primarily attributed to error-prone TLS across 6-4PPs, limited availability of dormant origins causes more frequent use of such mutagenic TLS to rescue 6-4PP-induced stalled forks. In turn, this finding suggests a promoting role of dormant origins in accurate bypass of 6-4PPs. To test this hypothesis, we propose the following specific aims: 1) Test if Mcm4c3/c3 cells rely more on TLS to bypass 6-4PPs at stalled forks, and 2) Determine the role of dormant origins in suppressing UV-induced triplet mutations and skin cancer. A novel aspect of this proposal is to test the anti-mutagenic role of dormant origins against residual 6-4PPs as a cellular defense mechanism to prevent the development of skin cancers.